The focus of this laboratory program is identification of inhibitors of serine/threonine protein kinases involved in early events in the raf-1 to MAP kinase pathway. Since these ubiquitous kinases are centrally involved in signaling that regulates cell growth and differentiation, such inhibitors are expected to have significant effects on the neoplastic cell phenotype. These kinases, which activate each other in a cascading pathway, signal downstream from oncogenic p21ras protein as well as downstream from protein kinase C (PKC) and tyrosine protein kinases (TPKs). The effect of this pathway is to phosphorylate and activate proteins involved in gene transcription and translation. This project evolved from a focus on modulation of PKC during the previous funding period. The shift in focus allows knowledge from recent discoveries in raf-MEK-MAPK signaling, as well as work done and experienced gained by the lab program leader to be applied to the targeting of this important signaling pathway for the drug discovery process. The specific goals of this projects are to: (1) identify inhibitors of p74raf-1, MEK, and ERK/MAP kinases; (2) characterize the specificity of these inhibitors; and (3) determine the efficacy of these inhibitors for blocking the activity of p74raf-1, MEK, or ERK/MAPK in cells, including acute mitogenic responses and growth of normal and transformed cells. Extracts, fractionation, compounds, and structure elucidation will be done by project 1. The screening method for raf-MEK- MAPK pathway inhibitors in this lab program is similar to that done for PKC, using purified recombinant enzymes and a radioactivity-based 96-well assay. However, these pathway kinases and their signaling roles were discovered more recently than PKC and changes in the screening and secondary assays are anticipated as more discoveries are made. Past experience indicates that prioritization of extracts for fractionation should assign low priority to samples containing inhibitory activity potentially caused by polyphenols, quinones, and flavinoids, which are common but uninteresting protein kinase inhibitors. Other factors that increase priority assignment for fractionation of extracts include high potency, availability, and high target specificity. Testing the specificity of isolated inhibitors includes testing for inhibition of PKC and PKA in this lab program, in addition to the assays are provided by projects 2 and 4. Acute in vitro cellular assays are done in this lab program and cytotoxicity testing and in vivo efficacy assays are done by Cores B and C, respectively. Inhibitors of these kinases will be unique and useful tools in signal transduction research and biochemically active compounds that are efficacious in cellular and whole animal rodent models will be candidates for preclinical development.